Purification of nucleic acids using metal-titanium oxides

ABSTRACT

The present disclosure relates to systems and methods for purifying nucleic acid. In particular, the present disclosure relates to systems and methods for purifying nucleic acids using metal or metal oxide compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/209,318, filed Jul. 13, 2016, which claims priority to U.S.Provisional Application Ser. No. 62/192,444 filed Jul. 14, 2015, theentirety of which is incorporated by reference herein.

TECHNICAL FIELD

The present disclosure relates to systems and methods for purifyingnucleic acid. In particular, the present disclosure relates to systemsand methods for purifying nucleic acids using metal or metal oxidecompositions.

BACKGROUND

Nucleic acids found in cells can be deoxyribonucleic acid or ribonucleicacid and can be genomic DNA, extrachromosomal DNA (e.g. plasmids andepisomes), mitochondrial DNA, messenger RNA, miRNA, and transfer RNA.Nucleic acids can also be foreign to the host and contaminate a cell asan infectious agent, e.g. bacteria, viruses, fungi or single celledorganisms and infecting multicellular organisms (parasites). Recently,detection and analysis of the presence of nucleic acids has becomeimportant for the identification of single nucleotide polymorphisms(SNPs), chromosomal rearrangements, the insertion of foreign genes, andalterations in methylation status of nucleic acids. These includeinfectious viruses, e.g. HIV and other retroviruses, jumping genes, e.g.transposons, and the identification of nucleic acids from recombinantlyengineered organisms containing foreign genes, e.g. Roundup Readyplants.

The analysis of nucleic acids has a wide array of uses. For example, thepresence of a foreign agent can be used as a medical diagnostic tool.The identification of the genetic makeup of cancerous tissues can alsobe used as a medical diagnostic tool, confirming that a tissue iscancerous, and determining the aggressive nature of the canceroustissue. Chromosomal rearrangements, SNPs and abnormal variations in geneexpression can be used as a medical diagnostic for particular diseasestates. Further, genetic information can be used to ascertain theeffectiveness of particular pharmaceutical drugs, known as the field ofpharmacogenomics.

While many nucleic acid purification procedures are well known and havebeen in existence for years, these procedures can be time consuming andmay employ reagents that present dangers to those performing thepurification. For example, it has long been known that DNA can readilybe obtained in a purified form from a test sample using organicextraction procedures, but such procedures can require severalextractions and therefore can be time consuming. Additionally, the useof some organic solvents is undesirable and dangerous if properprecautions are not followed.

Accordingly, there is a need for an efficient, effective and convenientmethod for isolating nucleic acids preparing cell-free nucleic acids foranalysis.

SUMMARY

The present disclosure relates to systems and methods for purifyingnucleic acid. In particular, the present disclosure relates to systemsand methods for purifying nucleic acids using metal or metal oxidecompositions.

For example, in some embodiments, the present disclosure provides amethod of capturing DNA and/or RNA from a biological sample, comprising:a) contacting the sample with a particle and/or solid support comprisingor coated with a metal or metal oxide such that DNA and/or RNA in thesample binds the particle or solid support; b) washing the particle orsolid support to remove contaminants; and c) eluting the DNA and/or RNAfrom the particle or solid support. In some embodiments, the metal oxideis CuTi. The present disclosure in not limited to particular amounts ofcopper and titanium. In some embodiments, the CuTi is present at a ratioof approximately 2:1 Cu to Ti (e.g., 3:1, 2:1, 1:1, 1:2, 1:3, etc.). Insome embodiments, the metal or metal oxide is AlTi, CaTi, CoTi, Fe₂Ti,Fe₃Ti, MgTi, MnTi, NiTi, SnTi, ZnTi, Fe₂O₃, Fe₃O₄, Mg, Mn, Sn, Ti, or Zn(e.g., anhydride or hydrated forms). In some embodiments, the particleshave a diameter of 0.3 to 2 μm (e.g., 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm,5.0 μm, 10.0 μm, 20.0 μm, 30.0 μm, 40.0 μm, 50.0 μm, etc.). In someembodiments, the particles or solid surface are magnetic (e.g.,paramagnetic, or ferromagnetic) (e.g. iron), metallic, an inorganicsolid (e.g., silica), a polymer, or a combination thereof. In someembodiments, the solid surface or particles have a planer, acicular,cuboidal, tubular, fibrous, columnar or amorphous shape. In someembodiments, the particles preferentially bind DNA (e.g. single ordouble stranded DNA) or RNA, depending on the metal or metal oxide. Insome embodiments, the method further comprises the step of eluting DNAand/or RNA from the particles. In some embodiments, the elutioncomprises an elution buffer. In some embodiments, the elution buffercomprises phosphate (e.g., an inorganic phosphate or an organophosphate)at a concentration of 0.5 to 20 mM (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 mM). In some embodiments, the elution buffer is at a concentrationhigher than 10 mM and is diluted to a lower concentration after thesample has been eluted. In some embodiments, the elution buffer is at aconcentration higher than 10 mM and a small volume is used in the assayto prevent inhibition. In some embodiments, the RNA and/or DNA is viral,eukaryotic, or prokaryotic RNA and/or DNA (e.g., from a pathogen). Insome embodiments, particles do not substantially bind DNA.

In some embodiments, the method further comprises the step ofdetermining the identity and/or amount of the RNA present in the sample(e.g., using one or more detection methods selected from, for example,amplification, hybridization, or sequencing).

Further embodiments provide systems and/or kits, comprising a) aparticle and/or solid support comprising or coated with a metal oxide;and b) an elution buffer. In some embodiments, kits further comprise oneor more reagents selected from, for example, one or more nucleic acidprimers and one or more nucleic acid probes, controls, instructions,buffers (e.g., binding and/or wash buffers), etc.

Additional embodiments provide a method of capturing RNA from abiological sample, comprising: a) contacting the sample with a particleand/or solid support comprising or coated with a metal oxide such thatRNA in the sample binds the particle but not DNA in the sample; b)washing the particle to remove contaminants; and c) eluting the RNA fromthe particle. In some embodiments, the particles bind less than 20%(e.g., less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of DNA in the sample. In someembodiments, the DNA is genomic, bacterial, and/or viral DNA.

Further embodiments provide the use of the kits or particles describedherein to purify RNA.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIGS. 1A-D shows statistical analysis of RNA binding by CuTi particles.

FIG. 2A-H shows signal from RNA binding by CuTi particles.

FIG. 3A-D shows RNA binding by Cu and CuTi particles.

FIG. 4A-D shows signal from RNA binding by CuTi particles.

FIG. 5A-B shows the binding of RNA to CuTi coated particles.

FIG. 6A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with buffers of different phosphateconcentrations.

FIG. 7A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with buffers of different phosphateconcentrations.

FIG. 8A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with buffers of different phosphateconcentrations.

FIG. 9A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with buffers of different phosphate concentrationsand ratios of Cu to Ti.

FIG. 10A-B shows the binding of RNA to CuTi coated particles.

FIG. 11A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with different ratios of Cu to Ti.

FIG. 12A-B shows the binding of RNA to CuTi coated particles.

FIG. 13A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with different ratios of Cu to Ti.

FIG. 14A-B shows the binding of RNA to CuTi coated particles.

FIG. 15A-D shows the results of one way analysis of binding of RNA toCuTi coated particles with different sized particles.

FIG. 16A-B shows the binding of RNA to CuTi coated particles.

FIG. 17A-D shows the results of one way analysis of elution of RNA fromCuTi coated particles with buffers of different phosphateconcentrations.

FIG. 18A-B shows the binding of RNA to CuTi coated particles.

FIG. 19A-D shows a comparison of the binding of RNA and DNA to CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 20A-B shows the binding of RNA to CuTi coated particles.

FIG. 21A-D shows a comparison of the binding of RNA and viral DNA toCuTi coated particles, FeO₃ particles, and silica particles.

FIG. 22 shows recovery of RNA and DNA from CuTi coated particlescompared to silica particles.

FIG. 23A-B shows the binding of RNA to CuTi coated particles.

FIG. 24A-B shows a comparison of the binding of RNA and genomic DNA fromCuTi coated particles, FeO₃ particles, and silica particles.

FIG. 25 shows recovery of RNA and DNA from CuTi coated particlescompared to silica particles.

FIG. 26A-B shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 27 shows the binding of RNA to CuTi coated particles.

FIG. 28A-B shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 29 shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 30A-B shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 31 shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 32A-B shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 33 shows a comparison of the elution of RNA and DNA from CuTicoated particles, FeO₃ particles, and silica particles.

FIG. 34A-B shows a comparison of the binding and elution of RNA and DNAfrom CuTi coated particles, FeO₃ particles, and silica particles.

FIG. 35 shows isolation of HCV with Fe₂O₃, CuTi and silica.

FIG. 36 shows isolation of RNA and DNA by CuTi particles.

FIG. 37A-F shows isolation of HBV DNA from CuTi and silica.

FIG. 38A-I shows isolation of HIV RNA from CuTi using different lysisbuffer conditions.

FIG. 39A-F shows HIV binding data for different metal particles.

FIG. 40A-F shows HBV binding data for different metal particles.

FIG. 41 shows recovery of HIV and HBV targets by metal particles.

FIG. 42A-F HIV shows binding data for different metal particles.

FIG. 43A-F shows HBV binding data for different metal particles.

FIG. 44 shows DNA and RNA binding by metal oxide and combinations ofmetal oxide-titanium oxide coatings on particles.

DETAILED DESCRIPTION

The present disclosure relates to systems and methods for purifyingnucleic acid. In particular, the present disclosure relates to systemsand methods for purifying nucleic acids using metal or metal oxidecompositions.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

As used herein, “a” or “an” or “the” can mean one or more than one. Forexample, “a” widget can mean one widget or a plurality of widgets.

A “blood-borne microorganism” is intended to encompass any microorganismthat can be found in blood. Examples of blood-borne microorganismsinclude bacteria, viruses, fungi, and parasites.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4 acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5(carboxyhydroxyl¬methyl) uracil, 5-fluorouracil, 5 bromouracil,5-carboxymethylaminomethyl 2 thiouracil, 5carboxymethyl¬aminomethyluracil, dihydrouracil, inosine, N6isopentenyladenine, 1 methyladenine, 1-methylpseudo¬uracil, 1methylguanine, 1 methylinosine, 2,2-dimethyl¬guanine, 2 methyladenine, 2methylguanine, 3-methyl¬cytosine, 5 methylcytosine, N6 methyladenine, 7methylguanine, 5 methylaminomethyluracil, 5-methoxy¬amino¬methyl 2thiouracil, beta D mannosylqueosine, 5′ methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio N6 isopentenyladenine, uracil 5 oxyaceticacid methylester, uracil 5 oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2 thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4thiouracil, 5-methyluracil, N-uracil 5 oxyacetic acid methylester,uracil 5 oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment is retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

The term “primer” refers to an oligonucleotide, whether occurringnaturally as in a purified restriction digest or produced synthetically,which is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (i.e., inthe presence of nucleotides and an inducing agent such as DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., as few as a single polynucleotide molecule),where the amplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR) are forms ofamplification. Amplification is not limited to the strict duplication ofthe starting molecule. For example, the generation of multiple cDNAmolecules from a limited amount of RNA in a sample using reversetranscription (RT)-PCR is a form of amplification. Furthermore, thegeneration of multiple RNA molecules from a single DNA molecule duringthe process of transcription is also a form of amplification.

As used herein, the term “particles” refers to a substrate or othersolid material that does not dissolve in aqueous solutions utilized innucleic acid purification or isolation. For example, in someembodiments, particles are substrates utilized in nucleic acidpurification and isolation. Examples include, but are not limited to,beads, spheres, or other shaped particles. In some embodiments,particles are coated or functionalized with material that enhancesnucleic acid binding (e.g., CuTi compounds).

As used herein, the terms “subject” and “patient” refer to any animal,such as a dog, a cat, a bird, livestock, and particularly a mammal, andpreferably a human.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a representative portion or cultureobtained from any source, including biological and environmentalsources. Biological samples may be obtained from animals (includinghumans) and encompass fluids, solids, tissues, and gases. Biologicalsamples include blood products, such as plasma, serum, and the like. Insome embodiments, samples comprise cells (e.g., tumor cells) or tissues(e.g., tumor or biopsy tissues) or nucleic acids (e.g., DNA or RNA)isolated from such cells or tissues. Environmental samples includeenvironmental material such as surface matter, soil, mud, sludge,biofilms, water, and industrial samples. Such examples are not howeverto be construed as limiting the sample types applicable to the presentdisclosure.

As used herein, the term “substantially bind” as in reference toparticles that do not substantially bind DNA, refers to particles thatbind DNA at a low level (e.g., relative to the level of RNA bound by theparticles). In some embodiments, particles that do not substantiallybind DNA have a higher affinity for RNA than DNA. For example, in someembodiments, particles bind less than 30%, 25%, 20%, 15%, 10%, or 5% asmuch DNA as RNA. In some embodiments, particles have a decreasedaffinity for DNA relative to RNA (e.g., decreased by 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more).

Embodiments of the Technology

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

Embodiments of the present disclosure provide metal (e.g., CuTi or otherTi particles) for use in purification of RNA. For example, in someembodiments, particles are used in the capture of RNA (e.g., frommicroorganism) present in biological samples. In some embodiments, thepresence of RNA is then detected using suitable methods (e.g., todetermine the presence, absence, or amount of microorganism (e.g., viraltarget)) in a biological sample.

Experiments described herein demonstrated that CuTi particles captureRNA as well as other methods but do not capture DNA as well as the othermethods. This means that the CuTi particles can selectively capture RNA.This is important, for example, in the measure method of RNA viruses. Insome embodiments, it is not desirable to capture DNA because thepresence of pro-viral DNA in the extraction could give an inaccuratedetermination of the amount of viral particles.

I. Capture

Embodiments of the present disclosure provide compositions and methodfor selectively capturing DNA or RNA. In some embodiments, compositionsand methods of the present disclosure utilize particles and/or solidsupports comprising or coated with metal oxides (See e.g., U.S. Pat. No.6,936,414; herein incorporated by reference in its entirety). Thepresent disclosure is not limited to particular metal oxides. In someembodiments, the metal oxide is a copper titanium oxide. In someembodiments, the CuTi is present at a ratio of approximately 2:1 Cu toTi (e.g., 3:1, 2:1, 1:1, 1:2, 1:3, etc.).

In some embodiments, the metal or metal oxide is AlTi, CaTi, CoTi,Fe₂Ti, Fe₃Ti, MgTi, MnTi, NiTi, SnTi, ZnTi, Fe₂O₃, Fe₃O₄, Mg, Mn, Sn,Ti, or Zn (e.g., an hydrated or hydrated forms).

In some embodiments, the particles have a diameter of 0.5 to 50 μm(e.g., 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 5.0 μm, 10.0 μm, 20.0 μm, 30.0μm, 40.0 μm, 50.0 μm, etc.). In some embodiments, particles and/or solidsurfaces are comprised of organic polymers such as polystyrene andderivatives thereof, polyacrylates and polymethacrylates, andderivatives thereof or polyurethanes, nylon, polyethylene,polypropylene, polybutylene, and copolymers of these materials. In someembodiments, particles are polysaccharides, in particular hydrogels suchas agarose, cellulose, dextran, Sephadex, Sephacryl, chitosan, inorganicmaterials such as e.g. glass or further metal oxides and metalloidoxides (in particular oxides of formula MeO, wherein Me is selectedfrom, e.g., Al, Ti, Zr, Si, B, in particular Al₂O₃, TiO₂, silica andboron oxide) or metal surfaces, e.g. gold.

In some embodiments, particles are magnetic (e.g., paramagnetic,ferrimagnetic, ferromagnetic or superparamagnetic).

In some embodiments, the particles and/or solid surface may have aplaner, acicular, cuboidal, tubular, fibrous, columnar or amorphousshape, although other geometries are specifically contemplated.

In some embodiments, commercially available particles (e.g., obtainedfrom ISK Magnetics, Valparaiso, Ind; Qiagen, Venlo, The Netherlands;Promega Corporation, Madison, Wis.; Life Technologies, Carlsbad, Calif.;Ademtech, NY, NY, and Sperotech, Lake Forest, Ill.).

In some embodiments, RNA capture comprises the step of contacting abiological sample (e.g., blood, blood product, cells, tissues, urine,semen, saliva, etc.) with a metal oxide particle. In some embodiments,the sample is processed prior to capture (e.g., cell lysis,purification, etc.). In some embodiments, the sample is not processed.

In some embodiments, particles described herein have the advantage ofnot substantially binding DNA (e.g., genomic, viral, and/or bacterialDNA) or not substantially binding RNA. For example in some embodiments,the particles bind less than 20% (e.g., less than 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) ofDNA or RNA in the sample.

After binding of DNA or RNA to the particle, particles are washed toremove unbound components of the sample (e.g., using a wash buffer). Insome embodiments, commercially available buffers (e.g., available fromQiagen, Venlo, The Netherlands; Promega Corporation, Madison, Wis.; andAbbott, Abbott Park, Ill.) are utilized. In some embodiments, particlesare then isolated from the sample (e.g., using a magnet, centrifugation,or other suitable technique such as those method described by theaforementioned commercial vendors).

In some embodiments, RNA and/or DNA is eluted from the particles orsolid support (e.g., using an elution buffer). In some embodiments, theelution buffer comprises phosphate (e.g., inorganic phosphate ororganophosphate) at a concentration of 1 to 10 mM (e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 mM). In some embodiments, the metal or metal oxideand/or phosphate concentration is chosen to preferentially bind and/orelute DNA or RNA (See e.g., Example 10 below).

In some embodiments, the present disclosure provides kits and systemsfor capturing and purifying DNA and/or RNA. In some embodiments, thekits and systems comprise the particles described herein, controls,buffers, instructions, solid supports, separation components, (e.g.,magnets), and the like. In some embodiments, kits and systems furthercomprising reagents for downstream analysis of captured RNA (e.g.,reagents for performing a detection assay described below).

In some embodiments, captured DNA or RNA is subjected to furtheranalysis to determine the identity and/or quantity of the DNA or RNA(e.g., using one or more detection methods described below). In someembodiments, the RNA is RNA from a pathogenic virus (e.g., bymoviruses,comoviruses, nepoviruses, nodaviruses, picornaviruses, potyviruses,sobemoviruses, luteoviruses (beet western yellows virus and potatoleafroll virus), the picorna like group (Picornavirata), carmoviruses,dianthoviruses, flaviviruses, pestiviruses, tombusviruses,single-stranded RNA bacteriophages, hepatitis C virus and a subset ofluteoviruses (barley yellow dwarf virus)—the flavi like group(Flavivirata), alphaviruses, carlaviruses, furoviruses, hordeiviruses,potexviruses, rubiviruses, tobraviruses, tricornaviruses, tymoviruses,apple chlorotic leaf spot virus, beet yellows virus and hepatitis Evirus—the alpha like group (Rubivirata), family Birnaviridae, familyChrysoviridae, family Cystoviridae, family Endornaviridae, familyHypoviridae, family Megabirnaviridae, family Partitiviridae, familyPicobirnaviridae, family Reoviridae—includes Rotavirus, familyTotiviridae, Botrytis porri RNA virus 1, Circulifer tenellus virus 1,Cucurbit yellows associated virus, Sclerotinia sclerotiorumdebilitation-associated virus, Spissistilus festinus virus 1, orderNidovirales, family Arteriviridae, family Coronaviridae—includesCoronavirus, SARS, family Mesoniviridae, family Roniviridae, orderPicornavirales, family Dicistroviridae, family Iflaviridae, familyMarnaviridae, family Picornaviridae—includes Poliovirus, Rhinovirus (acommon cold virus), Hepatitis A virus, family Secoviridae includessubfamily Comovirinae, genus Bacillariornavirus, genus Labyrnavirus,order Tymovirales, family Alphaflexiviridae, family Betaflexiviridae,family Gammaflexiviridae, family Tymoviridae, family Alphatetraviridae,family Alvernaviridae, family Astroviridae, family Barnaviridae, familyBromoviridae, family Caliciviridae—includes Norwalk virus, familyCarmotetraviridae, family Closteroviridae, family Flaviviridae—includesYellow fever virus, West Nile virus, Hepatitis C virus (HCV), Denguefever virus, family Leviviridae, family Luteoviridae—includes Barleyyellow dwarf virus, family Narnaviridae, family Nodaviridae, familyPermutotetraviridae, family Potyviridae, family Togaviridae—includesRubella virus, Ross River virus, Sindbis virus, Chikungunya virus,family Tombusviridae, family Virgaviridae, genus Benyvirus, genusBlunervirus, genus Cilevirus, genus Hepevirus—includes Hepatitis Evirus, genus Higrevirus, genus Idaeovirus, genus Negevirus, genusOurmiavirus, genus Polemovirus, genus Sobemovirus, genus Umbravirus,Acyrthosiphon pisum virus, Blueberry necrotic ring blotch virus,Botrytis virus F, Canine picodicistrovirus, Chronic bee paralysisassociated satellite virus, Extra small virus, Heterocapsacircularisquama RNA virus, Kelp fly virus, Le Blanc virus, Plasmoparahalstedii virus, Orsay virus, Rosellinia necatrix fusarivirus 1,Santeuil virus, Solenopsis invicta virus 2, Solenopsis invicta virus 3,order Mononegavirales, family Bornaviridae—Borna disease virus, familyFiloviridae—includes Ebola virus, Marburg virus, familyParamyxoviridae-includes Measles virus, Mumps virus, Nipah virus, Hendravirus, RSV and NDV, family Rhabdoviridae—includes Rabies virus, familyNyamiviridae—includes Nyavirus, family Arenaviridae—includes Lassavirus, family Bunyaviridae—includes Hantavirus, Crimean-Congohemorrhagic fever, family Ophioviridae, family Orthomyxoviridae—includesInfluenza viruses, genus Deltavirus—includes Hepatitis D virus, genusDichorhavirus, genus Emaravirus, genus Nyavirus—includes Nyamanini andMidway viruses, genus Tenuivirus, genus Varicosavirus, Taastrup virus,or Sclerotinia sclerotiorum negative-stranded RNA virus 1.

In some embodiments, particles find use in sensors that generate ormodulate an electrical signal upon the binding of a nucleic acid.

II. Assays

In some embodiments, following capture, RNA is detected and/orquantitated. Exemplary assays are described herein.

In some embodiments, assays are nucleic acid detection assays (e.g.,amplification, sequencing, hybridization, etc.). Illustrativenon-limiting examples of nucleic acid amplification techniques include,but are not limited to, polymerase chain reaction (PCR), reversetranscription polymerase chain reaction (RT-PCR), transcription-mediatedamplification (TMA), ligase chain reaction (LCR), strand displacementamplification (SDA), and nucleic acid sequence based amplification(NASBA). Those of ordinary skill in the art will recognize that certainamplification techniques (e.g., PCR) require that RNA be reversedtranscribed to DNA prior to amplification (e.g., RT-PCR), whereas otheramplification techniques directly amplify RNA (e.g., TMA and NASBA).

In some embodiments, nucleic acid sequencing methods are utilized (e.g.,for detection of amplified nucleic acids). In some embodiments, thetechnology provided herein finds use in a Second Generation (a.k.a. NextGeneration or Next-Gen), Third Generation (a.k.a. Next-Next-Gen), orFourth Generation (a.k.a. N3-Gen) sequencing technology including, butnot limited to, pyrosequencing, sequencing-by-ligation, single moleculesequencing, sequence-by-synthesis (SBS), semiconductor sequencing,massive parallel clonal, massive parallel single molecule SBS, massiveparallel single molecule real-time, massive parallel single moleculereal-time nanopore technology, etc. Morozova and Marra provide a reviewof some such technologies in Genomics, 92: 255 (2008), hereinincorporated by reference in its entirety. Those of ordinary skill inthe art will recognize that because RNA is less stable in the cell andmore prone to nuclease attack experimentally RNA is usually reversetranscribed to DNA before sequencing.

A number of DNA sequencing techniques are suitable, includingfluorescence-based sequencing methodologies (See, e.g., Birren et al.,Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; hereinincorporated by reference in its entirety). In some embodiments, thetechnology finds use in automated sequencing techniques understood inthat art. In some embodiments, the present technology finds use inparallel sequencing of partitioned amplicons (PCT Publication No:WO2006084132 to Kevin McKernan et al., herein incorporated by referencein its entirety). In some embodiments, the technology finds use in DNAsequencing by parallel oligonucleotide extension (See, e.g., U.S. Pat.No. 5,750,341 to Macevicz et al., and U.S. Pat. No. 6,306,597 toMacevicz et al., both of which are herein incorporated by reference intheir entireties). Additional examples of sequencing techniques in whichthe technology finds use include the Church polony technology (Mitra etal., 2003, Analytical Biochemistry 320, 55-65; Shendure et al., 2005Science 309, 1728-1732; U.S. Pat. Nos. 6,432,360, 6,485,944, 6,511,803;herein incorporated by reference in their entireties), the 454 picotiterpyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380;US 20050130173; herein incorporated by reference in their entireties),the Solexa single base addition technology (Bennett et al., 2005,Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; 6,833,246;herein incorporated by reference in their entireties), the Lynxmassively parallel signature sequencing technology (Brenner et al.(2000). Nat. Biotechnol. 18:630-634; U.S. Pat. Nos. 5,695,934;5,714,330; herein incorporated by reference in their entireties), andthe Adessi PCR colony technology (Adessi et al. (2000). Nucleic AcidRes. 28, E87; WO 00018957; herein incorporated by reference in itsentirety).

Next-generation sequencing (NGS) methods share the common feature ofmassively parallel, high-throughput strategies, with the goal of lowercosts in comparison to older sequencing methods (see, e.g., Voelkerdinget al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; each herein incorporated by reference in theirentirety). NGS methods can be broadly divided into those that typicallyuse template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), LifeTechnologies/Ion Torrent, the Solexa platform commercialized byIllumina, GnuBio, and the Supported Oligonucleotide Ligation andDetection (SOLiD) platform commercialized by Applied Biosystems.Non-amplification approaches, also known as single-molecule sequencing,are exemplified by the HeliScope platform commercialized by HelicosBioSciences, and emerging platforms commercialized by VisiGen, OxfordNanopore Technologies Ltd., and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. Nos.6,210,891; 6,258,568; each herein incorporated by reference in itsentirety), template DNA is fragmented, end-repaired, ligated toadaptors, and clonally amplified in-situ by capturing single templatemolecules with beads bearing oligonucleotides complementary to theadaptors. Each bead bearing a single template type is compartmentalizedinto a water-in-oil microvesicle, and the template is clonally amplifiedusing a technique referred to as emulsion PCR. The emulsion is disruptedafter amplification and beads are deposited into individual wells of apicotitre plate functioning as a flow cell during the sequencingreactions. Ordered, iterative introduction of each of the four dNTPreagents occurs in the flow cell in the presence of sequencing enzymesand luminescent reporter such as luciferase. In the event that anappropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 10⁶ sequence readscan be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. Nos. 6,833,246; 7,115,400; 6,969,488; each herein incorporated byreference in its entirety), sequencing data are produced in the form ofshorter-length reads. In this method, single-stranded fragmented DNA isend-repaired to generate 5′-phosphorylated blunt ends, followed byKlenow-mediated addition of a single A base to the 3′ end of thefragments. A-addition facilitates addition of T-overhang adaptoroligonucleotides, which are subsequently used to capture thetemplate-adaptor molecules on the surface of a flow cell that is studdedwith oligonucleotide anchors. The anchor is used as a PCR primer, butbecause of the length of the template and its proximity to other nearbyanchor oligonucleotides, extension by PCR results in the “arching over”of the molecule to hybridize with an adjacent anchor oligonucleotide toform a bridge structure on the surface of the flow cell. These loops ofDNA are denatured and cleaved. Forward strands are then sequenced withreversible dye terminators. The sequence of incorporated nucleotides isdetermined by detection of post-incorporation fluorescence, with eachfluor and block removed prior to the next cycle of dNTP addition.Sequence read length ranges from 36 nucleotides to over 250 nucleotides,with overall output exceeding 1 billion nucleotide pairs per analyticalrun.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. Nos. 5,912,148; 6,130,073; each hereinincorporated by reference in their entirety) also involves fragmentationof the template, ligation to oligonucleotide adaptors, attachment tobeads, and clonal amplification by emulsion PCR. Following this, beadsbearing template are immobilized on a derivatized surface of a glassflow-cell, and a primer complementary to the adaptor oligonucleotide isannealed. However, rather than utilizing this primer for 3′ extension,it is instead used to provide a 5′ phosphate group for ligation tointerrogation probes containing two probe-specific bases followed by 6degenerate bases and one of four fluorescent labels. In the SOLiDsystem, interrogation probes have 16 possible combinations of the twobases at the 3′ end of each probe, and one of four fluors at the 5′ end.Fluor color, and thus identity of each probe, corresponds to specifiedcolor-space coding schemes. Multiple rounds (usually 7) of probeannealing, ligation, and fluor detection are followed by denaturation,and then a second round of sequencing using a primer that is offset byone base relative to the initial primer. In this manner, the templatesequence can be computationally re-constructed, and template bases areinterrogated twice, resulting in increased accuracy. Sequence readlength averages 35 nucleotides, and overall output exceeds 4 billionbases per sequencing run.

In certain embodiments, the technology finds use in nanopore sequencing(see, e.g., Astier et al., J. Am. Chem. Soc. 2006 Feb. 8;128(5):1705-10, herein incorporated by reference). The theory behindnanopore sequencing has to do with what occurs when a nanopore isimmersed in a conducting fluid and a potential (voltage) is appliedacross it. Under these conditions a slight electric current due toconduction of ions through the nanopore can be observed, and the amountof current is exceedingly sensitive to the size of the nanopore. As eachbase of a nucleic acid passes through the nanopore, this causes a changein the magnitude of the current through the nanopore that is distinctfor each of the four bases, thereby allowing the sequence of the DNAmolecule to be determined.

In certain embodiments, the technology finds use in HeliScope by HelicosBioSciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. Nos.7,169,560; 7,282,337; 7,482,120; 7,501,245; 6,818,395; 6,911,345;7,501,245; each herein incorporated by reference in their entirety).Template DNA is fragmented and polyadenylated at the 3′ end, with thefinal adenosine bearing a fluorescent label. Denatured polyadenylatedtemplate fragments are ligated to poly(dT) oligonucleotides on thesurface of a flow cell. Initial physical locations of captured templatemolecules are recorded by a CCD camera, and then label is cleaved andwashed away. Sequencing is achieved by addition of polymerase and serialaddition of fluorescently-labeled dNTP reagents. Incorporation eventsresult in fluor signal corresponding to the dNTP, and signal is capturedby a CCD camera before each round of dNTP addition. Sequence read lengthranges from 25-50 nucleotides, with overall output exceeding 1 billionnucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on thedetection of hydrogen ions that are released during the polymerizationof DNA (see, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub.Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073,and 20100137143, incorporated by reference in their entireties for allpurposes). A microwell contains a template DNA strand to be sequenced.Beneath the layer of microwells is a hypersensitive ISFET ion sensor.All layers are contained within a CMOS semiconductor chip, similar tothat used in the electronics industry. When a dNTP is incorporated intothe growing complementary strand a hydrogen ion is released, whichtriggers a hypersensitive ion sensor. If homopolymer repeats are presentin the template sequence, multiple dNTP molecules will be incorporatedin a single cycle. This leads to a corresponding number of releasedhydrogens and a proportionally higher electronic signal. This technologydiffers from other sequencing technologies in that no modifiednucleotides or optics are used. The per-base accuracy of the Ion Torrentsequencer is ˜99.6% for 50 base reads, with ˜100 Mb to 100 Gb generatedper run. The read-length is 100-300 base pairs. The accuracy forhomopolymer repeats of 5 repeats in length is ˜98%. The benefits of ionsemiconductor sequencing are rapid sequencing speed and low upfront andoperating costs.

The technology finds use in another nucleic acid sequencing approachdeveloped by Stratos Genomics, Inc. and involves the use of Xpandomers.This sequencing process typically includes providing a daughter strandproduced by a template-directed synthesis. The daughter strand generallyincludes a plurality of subunits coupled in a sequence corresponding toa contiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Pat. Pub No. 20090035777, entitled “HighThroughput Nucleic Acid Sequencing by Expansion,” filed Jun. 19, 2008,which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. patentapplication Ser. No. 11/671,956; U.S. patent application Ser. No.11/781,166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectible fluorescence resonance energytransfer (FRET) upon nucleotide addition.

In some embodiments, detection methods utilize hybridization assays.Illustrative non-limiting examples of nucleic acid hybridizationtechniques include, but are not limited to, microarrays including, butnot limited to: DNA microarrays (e.g., cDNA microarrays andoligonucleotide microarrays). A DNA microarray, commonly known as genechip, DNA chip, or biochip, is a collection of microscopic DNA spotsattached to a solid surface (e.g., glass, plastic or silicon chip)forming an array for the purpose of expression profiling or monitoringexpression levels for thousands of genes simultaneously. The affixed DNAsegments are known as probes, thousands of which can be used in a singleDNA microarray. Microarrays can be used to identify disease genes ortranscripts by comparing gene expression in disease and normal cells.Microarrays can be fabricated using a variety of technologies, includingbut not limiting: printing with fine-pointed pins onto glass slides;photolithography using pre-made masks; photolithography using dynamicmicromirror devices; ink-jet printing; or, electrochemistry onmicroelectrode arrays.

Southern and Northern blotting is used to detect specific DNA or RNAsequences, respectively. DNA or RNA extracted from a sample isfragmented, electrophoretically separated on a matrix gel, andtransferred to a membrane filter. The filter bound DNA or RNA is subjectto hybridization with a labeled probe complementary to the sequence ofinterest. Hybridized probe bound to the filter is detected. A variant ofthe procedure is the reverse Northern blot, in which the substratenucleic acid that is affixed to the membrane is a collection of isolatedDNA fragments and the probe is RNA extracted from a tissue and labeled.

One illustrative detection method, the Hybridization Protection Assay(HPA) involves hybridizing a chemiluminescent oligonucleotide probe(e.g., an acridinium ester-labeled (AE) probe) to the target sequence,selectively hydrolyzing the chemiluminescent label present onunhybridized probe, and measuring the chemiluminescence produced fromthe remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283,174and Norman C. Nelson et al., Nonisotopic Probing, Blotting, andSequencing, ch. 17 (Larry J. Kricka ed., 2d ed. 1995, each of which isherein incorporated by reference in its entirety).

Attachment of fluorophores to nucleic acid probes is well known in theart and may be accomplished by any available means. Fluorophores can becovalently attached to a particular nucleotide, for example, and thelabeled nucleotide incorporated into the probe using standard techniquessuch as nick translation, random priming, PCR labeling, and the like.Alternatively, the fluorophore can be covalently attached via a linkerto the deoxycytidine nucleotides of the probe that have beentransaminated. Methods for labeling probes are described in U.S. Pat.No. 5,491,224 and Molecular Cytogenetics: Protocols and Applications(2002), Y.-S. Fan, Ed., Chapter 2, “Labeling Fluorescence In SituHybridization Probes for Genomic Targets,” L. Morrison et al., p. 21-40,Humana Press, both of which are herein incorporated by reference fortheir descriptions of labeling probes.

Exemplary fluorophores that can be used for labeling probes includeTEXAS RED (Molecular Probes, Inc., Eugene, Oreg.), CASCADE blueaectylazide (Molecular Probes, Inc., Eugene, Oreg.), SPECTRUMORANGE™(Abbott Molecular, Des Plaines, Ill.) and SPECTRUMGOLD™ (AbbottMolecular).

Examples of fluorophores that can be used in the methods describedherein are: 7-amino-4-methylcoumarin-3-acetic acid (AMCA); 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine B, 5-(and-6)-carboxyfluorescein; fluorescein-5-isothiocyanate (FITC);7-diethylaminocoumarin-3-carboxylic acid, tetramethyl-rhodamine-5-(and-6)-isothiocyanate; 5-(and -6)-carboxytetramethylrhodamine;7-hydroxy-coumarin-3-carboxylic acid; 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic acid; eosin-5-isothiocyanate;erythrosine-5-isothiocyanate; 5-(and -6)-carboxyrhodamine 6G; andCascades blue aectylazide (Molecular Probes, Inc., Eugene, Oreg.).

Probes can be viewed with a fluorescence microscope and an appropriatefilter for each fluorophore, or by using dual or triple band-pass filtersets to observe multiple fluorophores. See, e.g., U.S. Pat. No.5,776,688 to Bittner, et al., which is incorporated herein by reference.Any suitable microscopic imaging method can be used to visualize thehybridized probes, including automated digital imaging systems, such asthose available from MetaSystems or Applied Imaging. Alternatively,techniques such as flow cytometry can be used to examine thehybridization pattern of the chromosomal probes.

Probes can also be labeled indirectly, e.g., with biotin or digoxygeninby means well known in the art. However, secondary detection moleculesor further processing are then used to visualize the labeled probes. Forexample, a probe labeled with biotin can be detected by avidinconjugated to a detectable marker, e.g., a fluorophore. Additionally,avidin can be conjugated to an enzymatic marker such as alkalinephosphatase or horseradish peroxidase. Such enzymatic markers can bedetected in standard colorimetric reactions using a substrate for theenzyme. Substrates for alkaline phosphatase include5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.Diaminobenzoate can be used as a substrate for horseradish peroxidase.Fluorescence detection of a hybridized biotin or other indirect labeledprobe can be achieved by use of the commercially available tyramideamplification system.

Other agents or dyes can be used in lieu of fluorophores aslabel-containing moieties. Suitable labels that can be attached toprobes include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit luminescence, electrochemically activemolecules, enzymes, cofactors, and enzyme substrates. Luminescent agentsinclude, for example, radioluminescent, chemiluminescent,bioluminescent, and phosphorescent label containing moieties.Alternatively, detection moieties that are visualized by indirect meanscan be used. For example, probes can be labeled with biotin ordigoxygenin using routine methods known in the art, and then furtherprocessed for detection. Visualization of a biotin-containing probe canbe achieved via subsequent binding of avidin conjugated to a detectablemarker. The detectable marker may be a fluorophore, in which casevisualization and discrimination of probes may be achieved as describedabove for ISH.

In some embodiments, probes are designed to have labels placed at acommon interval throughout the nucleic acid (e.g., one label group every3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

In some embodiments, a probe library comprises probes with differentdetectable labels (e.g., different colors of fluorescent signal).

Probes hybridized to target regions may alternatively be visualized byenzymatic reactions of label moieties with suitable substrates for theproduction of insoluble color products. A biotin-containing probe withina set may be detected via subsequent incubation with avidin conjugatedto alkaline phosphatase (AP) or horseradish peroxidase (HRP) and asuitable substrate. 5-bromo-4-chloro-3-indolylphosphate and nitro bluetetrazolium (NBT) serve as substrates for alkaline phosphatase, whilediaminobenzidine serves as a substrate for HRP.

In embodiments where fluorophore-labeled probes or probe compositionsare used, the detection method can involve fluorescence microscopy, flowcytometry, or other means for determining probe hybridization. Anysuitable microscopic imaging method may be used in conjunction with themethods of the present invention for observing multiple fluorophores. Inthe case where fluorescence microscopy is employed, hybridized samplesmay be viewed under light suitable for excitation of each fluorophoreand with the use of an appropriate filter or filters. Automated digitalimaging systems such as the MetaSystems, BioView or Applied Imagingsystems may alternatively be used.

In some embodiments, the metal oxides described herein find use inbiosensors. For example, in some embodiments, the metal oxides are usedto coat electrical sensors that detect nucleic acids. In someembodiments, the metal oxide are used to capture nucleic acids (e.g., asdescribed above). Captured nucleic acids are then detected using atarget specific probe. In some embodiments, a target specific capturesequence is attached to the metal oxide and used to capture a specificnucleic acid target. In either case the binding of the nucleic acids tothe metal oxide generates a detectible signal.

EXPERIMENTAL Example 1

This example describes synthesis of CuTi coated particles. Differentamounts of copper and/or titanium were compared. Experiments were alsoconducted to determine if phosphate is needed for the metalprecipitation on the particles. NaOH is added first. The neutralizationitself should precipitate the metals. The metal oxides are insoluble aswell as the metal phosphates. The phosphate may not be precipitating anymetals at this point since they are already precipitated.

Reagents Common Name Vendor Copper(II) chloride Sigma-Aldrich HCl 12MSigma-Aldrich Iron Oxide-black Rockwood Potassium phosphate dibasicSigma-Aldrich Sodium Hydroxide 50% Sigma-Aldrich Sodium Hydroxide 5NFisher Sodium Phosphate dibasic Sigma-Aldrich Titanium(III) chlorideSigma-Aldrich solution

Twenty ml of 1 M CuC12 and 1 liter of 10 mM NaOH were prepared.

Particles were made using reagents below.

Tube 1 2 3 4 ml Ti 0.90 0.90 0.90 0.00 ml Cu 1.4 1.4 1.4 1.4 mlphosphate 3.6 1.8 0 0 ml NaOH 1.2 1.2 1.2 1.2 ml 12M HCl 0 0 0 0.05 mMTi 9.45 9.45 9.45 0 mM Cu 14 14 14 14 mM phosphate 18 9 0 0 mM NaOH229.2 229.2 229.2 229.2 mM HCl 0 0 0 6

Four aliquots of 10 g particles (Rockwood BK5000AP) were dispensed eachinto a 125 ml PETG bottle. 100 ml water was added to each bottle andbottles were placed on rotator to mix. Cu and Ti solutions were added toeach bottle, shaken vigorously and put on rotator. All the particlesuspensions were filtered through a 100 micron nylon filter.(Spectramesh #146488 (Spectrum Labs)). Fifty ml water was added tobottle after pouring over filter, shaken, and poured over filter tocombine. NaOH was added to bottles. Phosphate was added to bottles #1and #2, particles were washed, and captured particles magnetically.

Particles were washed 5 times with 100 ml water and resuspended up tototal volume of 100 ml with 10 mM NaOH. Particles were diluted to 1%with 10 mM NaOH.

Test Particles for RNA Extraction.

The below table shows particles that were tested.

mM mM total Cu Ti Cu NaOH PO4 CuTi cu/ti mixed filtered 6.615 10.5169.75 12.6 17.1 1.59 yes yes 12.6 18 291 24 30.6 1.43 yes yes 9.45 14232.8 18 23.5 1.48 yes yes 9.45 14 232.8 9 23.5 1.48 yes yes 9.45 14232.8 0 23.5 1.48 yes yes 0 14 232.8 0 14.0 * yes yesCSC extraction was performed. Elution buffer was diluted to 5 mMphosphate, 10 ml elution buffer and 30 ml water. IC was added to lysisbuffer-800 μl IC added to 70 ml LB. Particles were washed using LBwithout IC. Reagents were prepared by diluting elution buffer to 5 mMphosphate, 10 ml elution buffer and 30 ml water. Samples were prepared(HCV and Negative control) to a final dilution of 30 IU/ml.

Particle set up is shown below—each CSC run has multiple particle types(42 total samples).

Set up extraction cartridges. Six sets of 7

Lysis MMP Elution Buffer + as listed LB as 5 mM Loading IC below SampleWash 1 Wash2 phosphate temp Well 1-lysis 1.5 ml 100 μl 0.5 ml 50 C. Well2 Well 3 Well 4-Wash1 0.7 ml Well 5-Wash2A 0.8 ml Well 6-Wash2B 0.8 mlElution-5 mM 44 μl 73 C. phosphate

After amplification, the data was analyzed using MultiAnalyze. Resultsare shown in FIGS. 1A-D. Further statistical analysis was performedusing JMP. Results are shown in FIGS. 2-3. Statistical analysis (onewayanalysis of FAM-MR by mmp, oneway Analysis of FAM-Ct by mmp, onewayanalysis of VIC-Ct by mmp, oneway analysis of VIC-MR by mmp) are shownin FIGS. 2A-D.

Further data on additional particles (oneway analysis of FAM-Ct by MMP,oneway analysis of FAM-MR by MMP, and oneway analysis of VIC-MR by MMP)is shown in FIGS. 2E-F.

Results indicated that the particles made without titanium and onlycopper oxide did not perform as well as the other types. They did notpick up HCV well and the internal control signal is off by ˜3 CT (10fold difference).

A comparison of only the CuTi particles is shown in FIGS. 3A and B. VICMR is shown in FIG. 3C-D.

Further analysis (oneway analysis of FAM-Ct by MMP, oneway analysis ofFAM-MR by MMP, oneway analysis of VIC-Ct by MMP, and oneway analysis ofVIC-MR by MMP) is shown in FIGS. 4A-D.

Results show that the particles made without phosphate are not differentin their performance than the ones made with phosphate. Phosphate is notneeded in the production of these particles.

Example 2

This example describes an analysis of the ratio of Ti and Cu in metaloxide coatings of particles.

Reagents Common Name Vendor Copper(II) chloride Sigma-Aldrich HCl 12MSigma-Aldrich Iron Oxide-black Rockwood Potassium phosphate dibasicSigma-Aldrich Sodium Hydroxide 50% Sigma-Aldrich Sodium Hydroxide 5NFisher Sodium Phosphate dibasic Sigma-Aldrich Titanium(III) chlorideSigma-Aldrich solution

CuCl₂ prepared with HCl similar to the TiCl₃. One liter of 10 mM NaOH, 1liter of water, and 2 ml of 5N NaOH were prepared. The totalconcentration of Cu+Ti=24 mM. Ti was varied from 24 mM to 0 mM and Cuwas varied from 0 to 24 mM respectively.

The below tables shows the concentrations of Cu and Ti in the differentparticles generated.

Bottle 1 2 3 4 5 6 7 ml Ti 2.29 1.90 1.52 1.14 0.76 0.38 0.00 ml Cu 00.4 0.8 1.2 1.6 2 2.4 ml NaOH 3 3 3 3 3 3 3 mM Ti 24 20 16 12 8 4 0 mMCu 0 4 8 12 16 20 24 mM NaOH 582 582 582 582 582 582 582

Seven aliquots of 10 g particles (Rockwood BK5000AP) were dispensed intoan a 125 ml PETG bottle and 100 ml water was added to each bottle. Cuand Ti solutions were added to each bottle, shaken vigorously and put onrotator. No pre-mixing before adding metals was performed. All theparticle suspensions were filtered through a 100 micron nylon filter(Spectramesh #146488 (Spectrum Labs)). Fifty ml water was added tobottle after pouring over filter, shaken, and poured over filter tocombine. NaOH was added to bottles. The particles were washed with 10 mMNaOH prior to resuspending to 10% in 10 mM NaOH. This makes the finalconcentration closer to 10 mM NaOH for storage. Particles were capturedmagnetically, fluid was decanted, and ˜100 ml water added (this step wasrepeated 5-8 times). Particles were re-suspended up to total volume of100 ml with 10 mM NaOH and then diluted to 1% with 10 mM NaOH.

Test Particles for RNA Extraction.

Extraction testing was performed using elution with 0, 2.5 and 5 mMphosphate. CSC extraction was performed using elution buffer diluted to5 mM phosphate, 5 ml elution buffer and 15 ml water; elution bufferdiluted to 2.5 mM phosphate, 2.5 ml elution buffer and 17.5 ml water,and elution buffer 0 mM phosphate-water. IC was added to lysis buffer(800 μl IC added to 70 ml LB). Wash 1 was conducted using LB without IC.

Samples were prepared using HCV at a final concentration of 45 IU/ml anda negative control.

Each CSC run has multiple particle types as shown in the Table below.

Lysis MMP Elution Buffer + as listed LB as 5 mM Loading IC below SampleWash1 Wash2 phosphate temp Well 1-lysis 1.5 ml 100 μl 0.5 ml 50 C. Well2 Well 3 Well 4-Wash1 0.7 ml Well 5-Wash2A 0.8 ml Well 6-Wash2B 0.8 mlElution-increased 45 μl 73 C. to 45

After extraction, HCV purification assays were performed using 30 μlsample 30 μl master mix to reflect desired sample input volume.

After amplification, the data was analyzed using MultiAnalyze. Resultsare shown in FIGS. 5A-B. Further statistical analysis was performedusing JMP. Results of oneway Analysis of FAM-Ct by Sample ID (FIG. 6A),FAM-MR By Sample ID (FIG. 6B), VIC-Ct By Sample ID (FIG. 6C), and VIC-MRBy Sample ID (FIG. 6D) are shown.

The 0 mM phosphate had poor recovery for all the particles. Results showthat the phosphate is needed to elute the RNA. In conclusion, these datademonstrate that phosphate is needed for optimum elution of RNA fromCuTi particles.

Titanium Level and Phosphate Elution Concentration

No target signal with 0 mM phosphate. Results of oneway Analysis ofFAM-Ct By Ti-phos (FIG. 7A), FAM-MR By Ti-phos (FIG. 7B), VIC-Ct ByTi-phos (FIG. 7C), and VIC-MR By Ti-phos (FIG. 7D) are shown.

Results show that the 2.5 mM does not elute as well as the 5 mMphosphate

Results of oneway Analysis of FAM-Ct By Ti-phos (FIG. 8A), FAM-MR ByTi-phos (FIG. 8B), VIC-Ct By Ti-phos (FIG. 8C), and VIC-MR By Ti-phos(FIG. 8D) are shown. Results indicate that the 5 mM phosphate elutionshowed the Cu—Ti combination works better than the 100% Cu or the 100%Ti.

Results of oneway analysis of FAM-Ct by % Ti, oneway analysis of FAM-MRby % Ti, oneway analysis of VIC-Ct by % Ti, and oneway analysis ofVIC-MR by % Ti are shown in FIGS. 9A-D.

The 5 mM phosphate elution showed that the Cu—Ti combination worksbetter than the 100% Cu or the 100% Ti. The 33% Ti, 66% Cu works thebest, most notably seen with the internal control.

Example 3

This example describes further analysis of Cu—Ti ratios.

The total concentration of Cu+Ti was 24 mM. Ti was varied from 11 mM to5 mM and Cu was varied from 13 to 19 mM, respectively.

Reagents Common Name Vendor Copper(II) chloride Sigma-Aldrich HCl 12MSigma-Aldrich Iron Oxide-black Rockwood Potassium phosphate dibasicSigma-Aldrich Sodium Hydroxide 50% Sigma-Aldrich Sodium Hydroxide 5NFisher Sodium Phosphate dibasic Sigma-Aldrich Titanium(III) chlorideSigma-Aldrich solution

CuCl₂ was prepared with HCl similar to the TiCl₃. One liter of 10 mMNaOH was prepared. Cu—Ti solutions were prepared by mixing CuCl₂ andTiCl₃ into single tubes prior to adding to particles. NaOH was addedafter particles are filtered. The below table shows the concentration ofCu and Ti in the different samples tested.

Bottle 1 2 3 4 5 6 ml Ti 1.05 0.95 0.86 0.67 0.57 0.48 ml Cu 1.3 1.4 1.51.7 1.8 1.9 ml NaOH 3 3 3 3 3 3 mM Ti 11 10 9 7 6 5 mM Cu 13 14 15 17 1819 mM NaOH 582 582 582 582 582 582

Particles were prepared by weighing out 6 aliquots of 10 g particles(Rockwood BK5000AP), dispensing each into a 125 ml PETG bottle, andadding 100 ml water to each bottle. The Cu—Ti solution was added to eachbottle, shaken vigorously and put on rotator. All the particlesuspensions were filtered through a 100 micron nylon filter (Spectramesh#146488 (Spectrum Labs)). Fifty ml water was added to each bottle afterpouring over filter, shaking, and poured over filter to combine.Particles were returned to a clean PETG bottle. Three ml of 50% NaOH wasadded to each bottle of particles and bottles were placed on therotator. Particles were captured magnetically, the fluid was decanted,and particle were washed 5 time with 100 ml water. A 6th Wash wasperformed with ˜100 ml of 10 mM NaOH. The fluid was decanted andparticles were re-suspended to total volume of 100 ml 10 mM NaOH.

The particles were tested for RNA binding. Four mM phosphate was usedfor elution. IC was in the lysis buffer. Samples were prepared at ˜3×LOD(final concentration of HCV was 45 IU/ml)

Extractions were performed as described in the below table.

Lysis MMP Elution Buffer + as listed LB as 4 mM Loading IC below SampleWash1 Wash2 phosphate temp Well 1-lysis 1.5 ml 100 ul 0.5 ml 50 C. Well2 Well 3 Well 4-Wash1 0.7 ml Well 5-Wash2A 0.8 ml Well 6-Wash2B 0.8 mlElution-43 ul 43 ul 73 C.

After amplification, data was analyzed by MultiAnalyze (FIGS. 10A-B) andJMP. Oneway analysis of FAM-Ct by mMCu:mMTi, oneway analysis of FAM-MRby mMCu:mMTi, oneway analysis of VIC-Ct by mMCu:mMTi, and onewayanalysis of VIC-MR by mMCu:mMTi is shown in FIGS. 11A-D.

The FAM signals are not significantly different, which is due to the lowtiter of the samples and the variability of the assay at that level. Thelowest overall CT value is at the Cu:Ti ratio of 16 mM Cu to 8 mM Ti or2:1. These particles also show the lowest CT value for the internalcontrol.

Example 4

This example describes an analysis of the overall amount of precipitateusing the 16:08 Cu:Ti ratio.

Reagents Common Name Vendor Copper(II) chloride Sigma-Aldrich HCl 12MSigma-Aldrich Iron Oxide-black Rockwood Potassium phosphate dibasicSigma-Aldrich Sodium Hydroxide 50% Sigma-Aldrich Sodium Hydroxide 5NFisher Sodium Phosphate dibasic Sigma-Aldrich Titanium(III) chlorideSigma-Aldrich solution

CuCl₂ was prepared with HCl similar to the TiCl₃. Eight ml of the CuCl₂and 3.8 ml TiCl₃ solution were prepared. The below Table shows theamount of Cu and Ti in each sample.

Bottle 1 2 3 4 5 6 ml Cu—Ti 2.12 1.89 1.65 1.42 1.18 0.95 ml NaOH 2.72.4 2.1 1.8 1.5 1.2 mM Ti 7.170518 6.392585 5.590975 4.792747 3.994523.196292 mM Cu 14.15948 12.62331 11.04039 9.464143 7.887899 6.311655 mMNaOH 523.8 465.6 407.4 349.2 291 232.8 mM CuTi 21.33 19.01589 16.6313614.25689 11.88242 9.507947 100% 90% 80% 70% 60% 50% 40% % original 2421.6 19.2 16.8 14.4 12 9.6 mM Cu

Particles were prepared by weighing out 6 aliquots of 10 g particles(Rockwood BK5000AP), dispensing each into a 125 ml PETG bottle, andadding 100 ml water to each bottle. Cu—Ti solution was added to eachbottle, shaken vigorously, and put on rotator. All the particlesuspensions were filtered through a 100 micron nylon filter (Spectramesh#146488 (Spectrum Labs). Fifty ml water to was added to bottle afterpouring over filter, shaking, and pouring over filter to combine.Particles were returned to a clean PETG bottle. The calculated amount of50% NaOH was added to each bottle of particles. Bottles were place allon rotator. Particles were magnetically captured, the fluid wasdecanted, and particles were washed 5 times with 100 ml water. A 6thWash with ˜100 ml of 10 mM NaOH was performed. The fluid was decantedand particles were resuspended to a total volume of 100 ml in 10 mM NaOH

The particles were tested for RNA elution using 5 mM phosphate forelution. Reagents were prepared as described above. IC was in the lysisbuffer. Samples were prepared at ˜3×LOD (final concentration of HCV was45 IU/ml).

Extractions were performed as described in the Table below.

Lysis MMP Elution Buffer + as listed LB as 5 mM Loading IC below SampleWash1 Wash2 phosphate temp Well 1-lysis 1.5 ml 100 ul 0.5 ml 50 C. Well2 Well 3 Well 4-Wash1 0.7 ml Well 5-Wash2A 0.8 ml Well 6-Wash2B 0.8 mlElution-45 ul 45 ul 73 C.

After amplification, data was analyzed using MultiAnalyze. (FIGS. 12A-B)and JMP. Oneway analysis of FAM-Ct by mM CuTi, oneway analysis of FAM-MRby mM CuTi, oneway analysis of VIC-Ct by mM CuTi, and oneway analysis ofVIC-MR by mM CuTi is shown in FIGS. 13A-D.

Results showed that the FAM signals were not significantly different,which is due to the low titer of the samples and the variability of theassay at that level. The optimum IC value was with particles with 14.4mM CuTi.

Example 5

This example describes an analysis of particles made with 17 mM and 14mM CuTi against particles made with 24 mM CuTi. The Table below showssamples tested.

Lysis MMP Elution Buffer + as listed LB as 5 mM Loading IC below SampleWash1 Wash2 phosphate temp Well 1-lysis 1.5 ml 100 ul 0.5 ml 50 C. Well2 Well 3 Well 4-Wash1 0.7 ml Well 5-Wash2A 0.8 ml Well 6-Wash2B 0.8 mlElution-45 ul 45 ul 73 C.

HCV capture was performed. After amplification, data was analyzed usingMultiAnalyze (FIG. 14A-B) and JMP. Oneway analysis of FAM-Ct by mMCuTi,oneway analysis of FAM-MR by mMCuTi, oneway analysis of VIC-Ct bymMCuTi, and oneway analysis of VIC-MR by mMCuTi is shown in FIGS. 15A-D.

For the FAM signal, there was no significant difference between thethree particle batches. For the VIC signal, there was no significantdifference between the three particle batches for the VIC CT but the 14and 17 mM CuTi particles have a higher MR than the 24 mM CuTi particles.

Phosphate Concentration and Elution.

Experiments were performed to test the amount of phosphate needed toelute target from particles. Dilutions of elution buffer were made usingwater. Extractions and HCV capture assays were performed as describedabove. Data was analyzed using Multianalyze (FIG. 16A-B) and JMP. Onewayanalysis of FAM-Ct by Sample ID, oneway analysis of FAM-MR by Sample ID,oneway analysis of VIC-Ct by Sample ID, and oneway analysis of VIC-MR bySample ID is shown in FIGS. 17A-D.

Results shown that the FAM and VIC CT and MR are identical at 4, 5, and6 mM phosphate.

Example 6

This example describes analysis of how well the CuTi coated particlesbind DNA and RNA. HBV DNA and HCV RNA were used as the targets. Resultswere compared with iron oxide and silica particles. All samples wereeluted using 5.7 mM phosphate buffer.

HBV Extraction and Assay

Target HBV CalB 6.6 log IU/ml and IC at 36 μl per sample (10×concentration) were used. Extractions were done with a 58° C. lysistemperature.

After amplification, data was analyzed using MultiAnalyze (FIG. 18A-B)and JMP for analysis. Oneway analysis of FAM-Ct by method, onewayanalysis of FAM-MR by method, oneway analysis of VIC-Ct by method, andoneway analysis of VIC-MR by method is shown in FIGS. 19A-D.

Results show that the HBV DNA from the CuTi particles has a CT valuethat is almost 7 CTs higher than the silica method. This representsapproximately a 100 fold difference (=2{circumflex over ( )}6.7). EachCT represents a 2 fold difference.

The Fe₂O₃ method has a 1.7 CT difference which represents over a 3 folddifference. The Fe₂O₃ captures 33% of the DNA compared to the Silicamethod and the CuTi captures 1% of the HBV DNA.

For the internal control, the CuTi method has a CT value that is 5.5 CThigher than the silica method. This represents approximately a 50 folddifference (=2{circumflex over ( )}5.5). Again, one sample had noreading and essentially no recovery, so the recovery is even less than2%.

The Fe₂O₃ method has a 1.5 CT difference which again represents over a 3fold difference. The Fe₂O₃ captures 33% of the DNA compared to theSilica method and the CuTi captures less than 2% of the internal controlDNA.

The extractions were repeated as above except that an HCV sample wasprocessed at 100 IU/ml along with a standard amount of HCV internalcontrol.

HCV AT was diluted to first to 1000 IU/ml and then to 100 IU/ml usingnegative diluent. Setup was as above except that 17.1 μl of HCV internalcontrol was added to each lyis chamber.

Results of FAM analysis are shown in FIGS. 20A-B. Oneway Analysis ofFAM-Ct by method, oneway analysis of FAM-MR by method, oneway analysisof VIC-Ct by method, and oneway analysis of VIC-MR by method is shown inFIGS. 21A-D.

For the HCV FAM signal, the CuTi particles and the silica particles hadessentially identical CT and MR values. The Fe₂O₃ particles had a CTvalue just slightly above the other conditions that would be 40% lessthan the other signals. The MR values are not significantly differentfor all three conditions.

For the Internal control signal, the CuTi particles and the Fe₂O₃particles matched CT values and the silica particles had a slightlyelevated CT value which would be less than a 30% difference.

The CuTI particles demonstrate RNA recovery at least as good as theFe₂O₃ and the silica particles for both HCV RNA and the internal controlRNA (pumpkin).

Overall Summary:

The CuTi particles capture RNA as well as other methods but do notcapture DNA as well as the other methods. This means that the CuTiparticles can selectively capture RNA. This is important in the measuremethod of RNA viruses. It is not desirable to capture DNA because thepresence of pro-viral DNA in the extraction could give an inaccuratedetermination of the amount of viral particles.

FIG. 22 and the Table below shows CuTi recovery compared to silica.

% Recovery Target Silica CuTi HBV DNA 100  1 Pumpkin DNA 100  2 HCV RNA100  95 Pumpkin RNA 100 129

Example 7

This example describes analysis of how well the CuTi particles bindgenomic DNA. Genomic DNA, HBV DNA and HCV RNA were used as the targets.Results were compared with the iron oxide and total nucleic acid method(silica particles).

Some samples were re-eluted to test a heated wash step. Samples wereeluted with water (heated wash simulation) and then eluted withphosphate for the Fe₂O₃ and CuTi method. No 2^(nd) elution was performedfor the TNA-Silica method.

Targets were made using HBV CalB, HCV CalB, and genomic DNA. The firstextraction was CuTi particles, the elution tubes were removed andreplaced with blanks), the particles were manually captured and returnedto the automated sample preparation instrument. The second elution wasperformed and particles were resuspended particles by pipetting. Theywere placed back in the heater block, incubated 10 minutes and thenparticles were manually captured. All extraction were done with 176 μlelution and run with the 3 assays, HCV, HBV, and MYD88 genomic DNA.

Extraction from Fe₂O₃ particles was performed as above except that theelution is a two stage elution with 50 μl 20 mM phosphate followed by126 μl water.

Silica TNA extraction was performed using a single stage 176 μl waterelution. No second elution was performed.

After amplification, HBV data was analyzed using MultiAnalyze (FIGS.23A-B) and JMP. Oneway Analysis of FAM-Ct by Sample ID, and onewayanalysis of FAM-MR by Sample ID is shown in FIGS. 24A-B.

The Washing steps did not improve any of the HBV signals.

Next, the first phosphate elutions were compared with the silicaprocess. (FIG. 25). Oneway analysis of FAM-Ct by sample ID and onewayanalysis of FAM-MR by Sample ID is shown in FIGS. 26A-B.

The Fe₂O₃ process and the CuTi process do not isolate DNA as well as theTNA method (see Table below).

HBV FAM CT CT diff xfold % TNA CuTi 28.33 2.6  6.062866 16% Fe₂O₃ 27.071.34 2.531513 40% Silica 25.73

The Fe₂O₃ method isolated 40% of the HBV signal when processed withgenomic DNA. The CuTi method only isolated 16% DNA compared to thesilica particle TNA method.

The assay and analysis was repeated with HCV. FIG. 27 shows FAM resultsfor HCV. Oneway analysis of FAM-Ct by Sample ID and oneway Analysis ofFAM-MR by Sample ID is shown in FIGS. 29A-B. The Washing steps did notimprove any of the HBV signals.

The first phosphate elutions were compared with the silica process.(FIG. 29). Oneway analysis of FAM-Ct by Sample ID and oneway analysis ofFAM-MR by Sample ID is shown in FIGS. 30A-B. Results show that the Fe₂O₃method isolated more HCV RNA than either the CuTi method or the silicamethod in this experiment. The CuTi method isolated more RNA than thesilica TNA method. All three methods effectively isolate RNA.

Experiments were repeated with genomic DNA. FIG. 31 shows FAM results.Oneway analysis of CY5-Ct by Sample ID and oneway analysis of CY5-MR bySample ID is shown in FIGS. 32A-B.

Again, the Washing did not appear to improve the signals.

The first phosphate elutions were compared with the silica process (FIG.33). Oneway analysis of CY5-Ct by Sample ID and oneway analysis ofCY5-MR by Sample ID is shown in FIGS. 34A-B and the below Table.

MYD88 FAM CT CT diff xfold % TNA CuTi  35.666  3.086 8.491386 12% Fe2O334.22 1.64 3.116658 32% Silica 32.58

Results show that the Fe₂O₃ method isolates 32% of the genomic DNA. TheCuTi method only isolates 12% DNA compared to the silica particle TNAmethod.

SUMMARY

The Fe₂O₃ method isolates 40% of the HBV signal when processed withgenomic DNA. The CuTi method only isolates 16% DNA compared to thesilica particle TNA method.

The Fe₂O₃ method isolated more HCV RNA than either the CuTi method orthe silica method in this experiment. The CuTi isolated more RNA thanthe silica TNA method, 136%. All three methods effectively isolate RNAas can be seen by the amplification curves for FAM. There is a greatdeal of overlap in the curves and the CT values may not reflect thatsimilarity (FIG. 35).

The Fe₂O₃ method isolates 32% of the genomic DNA. The CuTi method onlyisolates 12% DNA compared to the silica particle TNA method.

The CuTi particles capture RNA as well as other methods but do notcapture DNA as well as the other methods. This means that the CuTiparticles can selectively capture RNA. This is important in the measuremethod of RNA viruses. It is not desirable to capture DNA because thepresence of pro-viral DNA in the extraction could give an inaccuratedetermination of the amount of viral particles (FIG. 36 and the Tablebelow shows CuTi recovery compared to Silica)

% Recovery Target Silica CuTi HBV DNA 100  16 Human DNA 100  12 HCV RNA100 136

Example 8

This example describes isolation of DNA using CuTi.

HBV was used at a concentration below LOD samples (10 cps/ml). For CuTiextractions, lysis buffer w/o ethanol for lysis and wash 1 were used,water was used for wash 2, and 5 mM Elution buffer was used. For thesilica extraction, lysis and wash 1 used 70 ml ethanol added to 70 mllysis buffer. For wash 2, 70 ml ethanol was added to 25 ml wash 2(water).

Samples were HBV at a final concentration of 10 IU/ml. The table belowshows the different samples tested and assay protocols.

8 samples 8 samples 8 samples 8 samples 20 ul IC 20 ul IC 20 ul IC 20 ulIC 1500 ul lysis 1500 ul lysis 500 ul lysis 500 ul lysis buffer totalbuffer total buffer total buffer total 50 ul PK 50 ul PK 50 ul PK 50 ulPK 150 ul LB 150 ul LB 150 ul LB 150 ul LB 200 ul sample 200 ul sample200 ul sample 200 ul sample 25 ul Silica MMP 25 ul Silica MMP 100 ulCuTi 100 ul CuTi 500 wash1 500 wash1 500 wash1 500 wash1 800 wash2A 800wash2A 800 wash2A 800 wash2A 800 wash2B 800 wash2B 800 wash2B 800 wash2B55 ul elution 55 ul elution 55 ul elution 55 ul elution 300 sec pk 300sec pk 300 sec pk 300 sec pk incubation incubation incubation incubation10 min lysis 10 min lysis 10 min lysis 10 min lysis 10 min elution 10min elution 10 min elution 10 min elution

After extractions were completed, assays were setup and run as above.After analysis, data transferred to MultiAnalyze and JMP as above.Results are shown in FIGS. 37A-F. Results show that all the samples at10 IU/ml were detected with both the CuTi particles and the silicaparticles. For example, 40/40 were detected for the CuTi particles and24/24 for the silica particles. In both experiments the target HBVsignals were statistically identical, although the MR for the CuTitrends higher than the silica particles. The internal control in thefirst experiment had a higher CT value for the CuTi particles but waslower than the silica particles in the second experiment. The HBV targetwas detected at below LOD at 100% detection with the CuTi particlepreparations and the internal control was also detected at levelscomparable to the silica process.

Example 9

This example describes DNA and RNA capture with CuTi particles.Different lysis buffer dilutions were tested to determine anydifferential DNA and RNA recovery. A total of 8 lysis bufferconcentrations were tested. HIV (1000 moleucles/ml) and HBV nucleicacids were tested. GITC is the primary component of the lysis buffer.The table below shows lysis buffer conditions.

Using 1 ml total lysis as lysis buffer and water-total vol is 1.37 mladd add MGITC lysis total IC sample lysis water lb MMP lysis vol voltotal GITC 0.02 1 0 0.2 1.3 0.1 1.566 1.3 2.62 0.777022901 0.02 1 0.2 01.3 0.1 1.566 1.5 2.62 0.896564885 0.02 1 0 0.2 1.3 0.1 2.35 1.3 2.621.166030534 0.02 1 0.2 0 1.3 0.1 2.35 1.5 2.62 1.345419847 0.02 1 0 0.21.3 0.1 3.13 1.3 2.62 1.553053435 0.02 1 0.2 0 1.3 0.1 3.13 1.5 2.621.791984733 0.02 1 0 0.2 1.3 0.1 4.7 1.3 2.62 2.332061069 0.02 1 0.2 01.3 0.1 4.7 1.5 2.62 2.690839695

Multianalyze5 was used to analyze data. Results are shown in FIG. 38A-I. The percent recovery was calculated using the lowest CT as themaximal value. Each CT higher represents ½ the recovery of the CT below(a Cycle Threshold is the difference from one amplification cycle to thenext, each cycle results in a doubling of the amount of amplicon.) TheCT differences were calculated and the percent recovery under each GITClevel (FIGS. 38H-I) was calculated for the RNA and the DNA recovery.

The DNA recovery is highest at high levels of GITC (2.33 M GITC andabove) while the RNA recovery is high at 1.35 M GITC and above. At 1.35M GITC using the extraction conditions described above (58° C., 1 mlsample and 1.5 ml lysis buffer) there is almost maximal RNA recovery butless than 10% DNA recovery for the RNA and DNA targets described above.

Example 10

This example describes DNA and RNA binding by metal particles. Thepurpose of this experiment was to expand the metal oxides tested for RNAand DNA binding beyond the CuTi particle formulation.

The following particles were prepared:

Metal Chlorides 1 AlCl3 2 Calcium chloride 1.0M 3 CoCl2 4 Chromium(III)chloride 5 Copper(II) chloride 6 FeCl2-Iron(II)chloride 7FeCl2-Iron(III)chloride 8 Manganese (II) Chloride 9 MgCL2 10 NiCl2 11SnCl2 12 Titanium(III) chloride solution 13 Zinc chloride

Particles were prepared in in 125 ml PETG bottles with 1 g particles.100 ml water was added to each. Bottle 1 had 0.5 ml of AlCl₃—HCl; Bottle2 had 0.5 ml of CaCL2+0.5 ml 3M HCl; Bottle 3 had 0.5 ml CoCl₂—HCl;Bottle 4 had 0.5 ml CuCl₂—HCl. Bottles were incubated on rotator for 50minutes. Particles were then neutralized with 0.63 ml 50% NaOH.

The particles were tested for HIV and HBV extraction using the followingreagents.

Extraction Reagents Lysis buffer Wash2 Water Elution buffer (20 mMphosphate) Elution buffer (5 mM phosphate) Neg Diluent HBV IC HIV IC TheHIV IC (17 ul) was added directly to the lysis well during processingThe HBV IC was added directly to the master mix prior to running theassay. No sample prep on the HBV IC, to look for assay inhibitiondirectly.Particles (100 μl) are added manually to the lysis well prior tostarting extraction.

Bottle 1-Al oxide ppt Bottle 5-Fe2 oxide ppt Bottle 9-Ni oxide pptBottle 2-CaCl oxide ppt Bottle 6-Fe3 oxide ppt Bottle 10-Sn oxide pptBottle 3-Cl oxide ppt Bottle 7-Mg oxide ppt Bottle 11-Ti oxide pptBottle 4-Cu oxide ppt Bottle 8-Mn oxide ppt Bottle 12-Zn oxide ppt

posi- samples tion module #1 module #2 module #3 #1 Bottle 1- Bottle9-Ni oxide ppt Bottle 5-Fe2 oxide ppt Al oxide ppt #2 Bottle 2- Bottle10-Sn oxide ppt Bottle 6-Fe3 oxide ppt CaCl oxide ppt #3 Bottle 3-Bottle 11-Ti oxide ppt Bottle 7-Mg oxide ppt Cl oxide ppt #4 Bottle 4-Bottle 12-Zn oxide ppt Bottle 8-Mn oxide ppt Cu oxide ppt #5 Bottle 5-Bottle 1-Al oxide ppt Bottle 9-Ni oxide ppt Fe2 oxide ppt #6 Bottle 6-Bottle 2-CaCl oxide ppt Bottle 10-Sn oxide ppt Fe3 oxide ppt #7 Bottle7- Bottle 3-Cl oxide ppt Bottle 11-Ti oxide ppt Mg oxide ppt #8 Bottle8- Bottle 4-Cu oxide ppt Bottle 12-Zn oxide ppt Mn oxide ppt

The 2nd Extraction same as the first except that the elution buffer was20 mM phosphate undiluted and a two-step elution (25 ul of 20 mM then 75ul of water).

After extraction, particles were assayed for binding to HBV and HIVusing RealTime assays described above.

FIGS. 39A-F shows HIV data for the different particles. FIGS. 40A-F showHBV data for the different particles.

The different metal oxides show differential binding to HIV and HBV.Some of the eluates show inhibition in the reactions as can be seen inthe IC signal from HBV. Nickel and Cobalt oxides show inhibition. Cu andFez also show inhibition. To compare the relative recovery of RNA andDNA, the cycle threshold values (CT) were used to calculate the relativerecovery of the targets to the particles that gave the best recovery.For example, if one oxide had a CT of 20, and another had a CT value of21, then the second oxide recovered ½ the amount of the first. Ifanother had a CT value of 22, then it only recovered ¼ the amount of thefirst. The calculation is CT difference from the lowest value (bestrecovery) which is then used as the exponent to 2^(X)

diff max % Recovery HBV Al-05 2 30.455 3.43 10.77787  9% Al-20 2 30.453.43 10.74058  9% Ca-05 2 30.135 3.11 8.633826 12% Ca-20 2 30.045 3.028.111676 12% Co-05 2 36.825 9.80 891.4438  0% Co-20 2 35.91 8.89472.7717  0% Cu-05 1 32.24 5.22 37.14253  3% Cu-20 2 31.055 4.0316.33619  6% Fe2-05 2 28.74 1.72 3.282966 30% Fe2-20 2 30.065 3.048.224911 12% Fe3-05 2 27.865 0.84 1.79005 56% Fe3-20 2 27.59 0.571.479388 68% Mg-05 2 32.31 5.29 38.98913  3% Mg-20 2 32.105 5.0833.82458  3% Mn-05 2 27.265 0.24 1.180993 85% Mn-20 2 27.025 0.00 1100%  Ni-05 2 34.36 7.34 161.4563  1% Ni-20 2 36.48 9.46 701.8408  0%Sn-05 2 32.635 5.61 48.84029 2% Sn-20 2 32.76 5.74 53.26072  2% Ti-05 227.92 0.90 1.85961 54% Ti-20 2 27.575 0.55 1.464086 68% Zn-05 2 28.241.22 2.321408 43% Zn-20 2 28.5 1.48 2.779836 36% HIV Al-05 2 22.13 2.264.773343 21% Al-20 2 21.16 1.29 2.436821 41% Ca-05 2 21.725 1.853.605002 28% Ca-20 2 21.42 1.55 2.918041 34% Co-05 2 28.345 8.47 354.588 0% Co-20 2 26.07 6.20 73.26235  1% Cu-05 2 23.895 4.02 16.22335  6%Cu-20 2 22.62 2.75 6.703897 15% Fe2-05 2 21.66 1.79 3.446185 29% Fe2-202 21.585 1.71 3.271608 31% Fe3-05 2 21.405 1.53 2.887858 35% Fe3-20 220.46 0.59 1.500039 67% Mg-05 2 21.37 1.50 2.818642 35% Mg-20 2 21.091.22 2.321408 43% Mn-05 2 21.11 1.24 2.353813 42% Mn-20 2 20.825 0.951.931873 52% Ni-05 2 27.635 7.76 216.7668  0% Ni-20 2 28.445 8.57380.038  0% Sn-05 2 19.995 0.12 1.086735 92% Sn-20 2 20.34 0.47 1.38031772% Ti-05 2 20.8 0.93 1.898684 53% Ti-20 2 20.605 0.73 1.658639 60%Zn-05 2 19.875 0.00 1 100%  Zn-20 2 20.025 0.15 1.109569 90%

As can be seen FIG. 41, various metal oxides recover the HIV and HBVtargets to different degrees. The Al, Ca, Co, Cu, Fez, Mg and Ni oxideparticles do not recover either target as well as the other metaloxides. The Fe₃ and Ti oxides recover both RNA and DNA, the Mn oxidecoated particles recover more DNA than RNA, and the Sn and Zn oxideparticles recover more RNA than DNA. The lysis conditions are 58° C. andthe sample:lysis volume ratio of 1:1.5 and the phosphate elution was 5and 20 mM. Other temperatures and other sample volume ratios may changethe relative recoveries. The amount of phosphate used to elute thetargets may also have an effect on the target recovery.

Additional experiments were conducted to retest metal oxides andmetal-titanium oxides. The following particles were tested:

Mg, Mn, Sn, Ti, Zn oxide coated particles.

Fe₃O₄ particles

Al—Ti, Ca—Ti, Co—Ti, Cu—Ti, Fe₂—Ti, Fe₃—Ti, Mg—Ti, Mn—Ti, Ni—Ti, Sn—Ti,and Zn—Ti oxide coated particles.

Particles were prepared and tested as described above. FIGS. 42A-F showsHIV data. FIGS. 43A-F shows HBV data. The metal oxide and metaloxide-titanium oxide precipitate coated particles show differential RNAand DNA binding. To compare the relative recovery of RNA and DNA, thecycle threshold values (CT) were used to calculate the relative recoveryof the targets to the particles that gave the best recovery. As is shownin FIG. 44, particular metal oxide and combinations of metaloxide-titanium oxide coatings on the magnetic particles have differentDNA and RNA binding properties. All of the oxides tested in thisexperiment show some nucleic acid recovery. The best oxide coatings forthe purification of RNA under the tested conditions are Cu—Ti, Mg—Ti,Sn, and Zn oxides. The best oxide coatings for the purification of DNAunder the tested conditions are Fe₃—Ti and Mn oxides. The best oxidecoatings for the purification of both RNA and DNA under the testedconditions are Mn—Ti and Sn—Ti oxides. The recovery is dependent uponthe ability of the metal oxide to bind the nucleic acids under thetested conditions, retain the nucleic acids under the wash conditionsand also release the bound nucleic acids under the elution conditions.

HBV HIV % Recovery % Recovery oxide AlTi 41% 44% CaTi 59% 71% CoTi 26%60% CuTi 59% 88% RNA Fe2Ti 61% 33% Fe3Ti 77% 48% DNA MgTi 42% 100%  RNAMnTi 97% 79% DNA-RNA NiTi 10% 46% SnTi 88% 91% DNA-RNA ZnTi 26% 42%Fe2O3-mmp 39% 96% Fe3O4-mmp 73% 45% Mg  3% 36% Mn 100%  31% DNA Sn  3%79% RNA Ti 71% 55% Zn 43% 84% RNA

All patents, patent application publications, journal articles,textbooks, and other publications mentioned in the specification areindicative of the level of skill of those in the art to which thedisclosure pertains. All such publications are incorporated herein byreference to the same extent as if each individual publication werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of capturing RNA and/or DNA from abiological sample, comprising: a) contacting said sample with a particleor solid surface comprising or coated with a metal oxide such that DNAand/or RNA in said sample binds said particle wherein said metal oxideis selected from the group consisting of AlTi, CaTi, CoTi, Fe₂Ti, Fe₃Ti,MgTi, MnTi, NiTi, SnTi and ZnTi; b) washing said particle or solidsurface to remove contaminants; and c) eluting RNA and/or DNA from saidparticle or solid surface.
 2. The method of claim 1, wherein said metaloxide is an anhydride or hydrated form of said metal oxide.
 3. Themethod of claim 1, wherein said particle has a diameter of 0.5 to 50 μm.4. The method of claim 1, wherein said particle or solid surface iscomprised of a polymer, a magnetic material, a metallic material, aninorganic solid, or a combination thereof.
 5. The method of claim 4,wherein said inorganic solid is silica.
 6. The method of claim 1,wherein said solid surface has a shape selected from the groupconsisting of planer, acicular, cuboidal, tubular, fibrous, columnar,and amorphous.
 7. The method of claim 1, wherein said eluting comprisesan elution buffer.
 8. The method of claim 7, wherein said elution buffercomprises phosphate.
 9. The method of claim 8, wherein said phosphate isan inorganic or an organophosphate.
 10. The method of claim 8, whereinsaid phosphate is present in said elution buffer at a concentration of0.5 to 20 mM.
 11. The method of claim 1, wherein said RNA and/or DNA iseukaryotic, prokaryotic or viral RNA.
 12. The method of claim 11,wherein said DNA and/or RNA is from a eukaryotic, prokaryotic or viralpathogen.
 13. The method of claim 1, wherein said particle or solidsurface preferentially binds RNA or DNA.
 14. The method of claim 1,further comprising the step of determining the identity and/or amount ofsaid DNA and/or RNA present in said sample.
 15. The method of claim 14,wherein said determining comprises the use of one or more detectionmethods selected from the group consisting of amplification,hybridization, and sequencing.